Flat mount preparation for whole-mount fluorescent imaging of zebrafish embryos

The zebrafish is a widely used model organism for biomedical research due to its ease of maintenance, external fertilization of embryos, rapid embryonic development, and availability of established genetic tools. One notable advantage of using zebrafish is the transparency of the embryos, which enables high-resolution imaging of specific cells, tissues, and structures through the use of transgenic and knock-in lines. However, as the embryo develops, multiple layers of tissue wrap around the lipid-enriched yolk, which can create a challenge to image tissues located deep within the embryo. While various methods are available, such as two-photon imaging, cryosectioning, vibratome sectioning, and micro-surgery, each of these has limitations. In this study, we present a novel deyolking method that allows for high-quality imaging of tissues that are obscured by other tissues and the yolk. Embryos are lightly fixed in 1% PFA to remove the yolk without damaging embryonic tissues and are then refixed in 4% PFA and mounted on custom-made bridged slides. This method offers a simple way to prepare imaging samples that can be subjected to further preparation, such as immunostaining. Furthermore, the bridged slides described in this study can be used for imaging tissue and organ preparations from various model organisms.